In the following document of the present description, when nucleic-acid base is indicated, A represents adenine; C represents cytosine; G represents guanine; T represents thymine and U represents uracil; respectively. In addition, when “oligonucleotide” is mentioned, it includes “polynucleotide” in some cases.
Nontuberculous mycobacteria is a gram positive bacillus having acid-fast characteristics classified into genus Mycobacterium (hereinafter, optionally abbreviated simply as M.), and is a kind of acid-fast bacterium other than tuberculosis complex and Mycobacterium leprae. Fifteen to 20% of cases showing positive for sputum smear examination for acid-fast bacterium have been diagnosed thereafter to be nontuberculous mycobacterium by the test for the identification of bacterial species.
Among nontuberculous mycobacteria, clinically problematic bacterial species are known to be M. intracellulare, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium gordonae, Mycobacterium szulgai, Mycobacterium avium, Mycobacterium xenopi, Mycobacterium fortuitum, Mycobacterium chelonei, Mycobacterium abscessus, and so on.
Above all, the commonly noted strains are M. intracellulare and M. avium. Since M. intracellulare and M. avium are closely resemble each other and difficult to distinguish between them, M. intracellulare and M. avium have been referred to collectively as Mycobacterium avium complex (MAC). About 70% of patients with nontuberculous mycobacterial disease are MAC infection, and the second large population is M. kansasii infection accounting 20%. And the rest of 10% are the infection by other bacterial species.
In general, since the nontuberculous mycobacteria have weak toxicity, they are believed to be harmless to a healthy subject. However, on rare occasions, they may exert infectivity to human. Among them, the MAC is known to cause sometimes aftereffects of tuberculosis (lung infectious disease), or to cause opportunistic infections to a compromised patient such as AIDS patient. Therefore, it is particularly important in the therapy to detect the nontuberculous mycobacterium with rapidity and preciseness.
In addition, in recent years, the incidence of nontuberculous mycobacterial infection demonstrates upward trend, and therefore, development of a method for discriminating tuberculosis bacterium from nontuberculous mycobacterium in a short period of time has been desired strongly. Moreover, from the viewpoint of the fact that the method of detecting/diagnosing M. intracellulare and M. avium by nucleic acid amplification technology has been included in health insurance coverage, its diagnostic significance is obviously great.
In addition, most of the nontuberculous mycobacteria demonstrate resistance against antituberucular agents. Therefore, when a patient is suspected of acid-fast bacterium infection, differential diagnosis whether the disease is tuberculosis or nontuberculous mycobacterial disease is quite important to decide a course of treatment. Further, as a method for treatment of the diseases caused by nontuberculous mycobacterium may vary depending on the individual species of bacterium, the identification of bacterial species is also quite important. However, nontuberculous mycobacteral diseases do not show any specific clinical symptom. Therefore, it is quite difficult to differentiate tuberculosis from nontuberculous mycobacterial disease by clinical observation and histopathological manifestation, and to specify the species of the nontuberculous mycobacterium. Consequently, the diagnosis whether the disease is tuberculosis or nontuberculous mycobacterial disease has to be carried out by identification of the infected bacterium.
A typical method for the identification of bacterium to be carried out for the diagnosis of nontuberculous mycobacterial disease is sputum smear examination. However, by this test, it can be recognized only whether the pathogenic bacterium is “acid-fast bacterium-positive” or not, and cannot be identified whether the pathogenic bacterium is tuberculosis bacteria or nontuberculous mycobacterium. Therefore, when result of the sputum smear examination is positive, bacterial culture examination by isolation culture on a culture medium such as Ogawa's medium is carried out to differentiate between tuberculosis bacteria and nontuberculous mycobacterium. And further, by performing additional biochemical examinations, bacterial species of the infected bacterium is identified. However, in general, growth of genus Mycobacterium is slow; for example, it takes 3 to 4 weeks only for its isolation culture. And further, it requires additional 2 to 3 weeks to obtain results of various biochemical tests for the identification of bacterial species. Accordingly, the conventional basic method, in which a diagnostic outcome on whether the disease is tuberculosis or not is obtained by conducting the above described smear examination and a cell culture assay, is a considerably time-consuming method.
On the other hand, in recent years, a technology of detecting bacteria on a genetic level has been developed. For example, a diagnostic technique utilizing the nucleic acid amplification technology such as polymerase chain reaction (PCR) and the like has been studied as a useful means for detecting bacteria. Because of high sensitivity of this method, even if there are only several cells of the bacteria in a sample, the bacteria can be detected. In addition, this method has an advantage that the detection (identification of bacterial species) can be completed in a short time (in 4 days at the longest). However, in the usual PCR method, the number of bacteria cannot be determined. In addition, in this method, cells are detected regardless of live cells or dead cells. Further, if some bacteria exist in the sample, the determination is made positive regardless of size of the bacterial count. Therefore, by the PCR method, diagnosis of infectivity will be provided with uncertainty. Furthermore, the method has other problems such as that judgment of false positive tends to be made due to too high sensitivity.
With respect to the method for detection of M. intracellulare using the PCR method, there is a method for detection of existence or absence of MAC nucleic acid using a multiple primer set of oligonucleotide primer specific for 2 or more of gene regions comprising, for example, MacSequevar gene region, 19 kD protein (MAV 19k) gene region of M. avium, and ribosomal protein s1 gene region of M. intracellulare (Patent Literature 1). However, by this method, discrimination between M. intracellulare and M. avium cannot be achieved. In addition, in the PCR using the employed rps1 primer (a primer designed from the ribosomal protein s1 gene region of M. intracellulare), even when the isolated strain of M. avium is used as a sample, the amplification product has also been detected; there remains a problem in terms of the specificity for M. intracellulare. 
In addition, a method has also been known, in which PCR is performed by using a primer which is capable of amplifying a DNA nucleotide sequence targeting insertion site of the gene insertion sequence IS901, and determining whether it is avian tuberculosis bacterium (M. avium) or M. intracellulare based on a chain length of the obtained amplification product (Patent Literature 2). However, in the PCR using the aforementioned primer, the primer extension product can be obtained not only when the sample is avian tuberculosis bacterium (M. avium) but also M. intracellulare, and therefore, this determination method can not be said as a specific method for M. intracellulare. In addition, the method, whereby the discrimination between both bacterial species is carried out based on the chain length of the primer extension product, is cumbersome; and it is conceivable that different determination may be made depending on the judge; and in consequence, the method cannot be said as a reliable determination method.
Other than the PCR method, there is a determination method through the use of Strand Displacement Amplification Method (SDA method). For example, JP-A-10-4984 (Patent Literature 3) discloses a method in which the 63 nucleotide segment of BCG85-B gene coding a part of α-antigen of mycobacteria is targeted. In this method, using a primer which is capable of amplifying the target sequence in the BCG85-B gene owned by both M. intracellulare and M. avium, nucleic acid amplification reaction is performed by the SDA method, and then MAC is detected based on the results. That is, the primer used in the aforementioned method is a primer capable of amplifying both M. intracellulare and M. avium. However, in this method, as a matter of course, a primer extension product will be obtained in both cases where either of M. intracellulare or M. avium exists in a sample. Because of this, MAC can be detected by this method; however, it is impossible to detect M. intracellulare specifically. In addition, even when MAC is detected, there can be an instance where false-positive result is provided.
In JP-A-2001-103986 (Patent Literature 4), a primer to be used for the detection of MAC and an oligonucleotide to be used as a capture probe and a detection probe have been disclosed. However, the aforementioned primer can amplify a 48 bp target sequence from dnaJ gene which is owned commonly by both M. intracellulare and avian tuberculosis bacteria (M. avium). Namely, amplification reaction will take place in both cases where either of M. intracellulare or M. avium is present in a sample. Therefore, if the SDA method is practiced using aforementioned primer, the primer extension product will be detected using the capture probe and detection probe, and based on the results, detection of MAC can be achieved. However, specific detection of M. intracellulare is impossible to achieve without detection of M. avium. 
Beyond that, there is a method of amplification of nucleic acid of M. intracellulare through the use of LAMP (Loop-Mediated Isothermal Amplification) method, and the like. However, in the LAMP method, there remains some problems such as that the nucleotide sequence of amplified DNA cannot be determined; that efficient length of DNA to be amplified is limited; and that the method provides false-positive result occasionally; and the like.
To solve these problems, the present inventor have already applied PCT/JP2007/059251 for patent (Patent Literature 6). In this application, a primer and a probe which were developed focusing on the capability of distinguishing/detecting in distinction of M. intracellulare from M. avium have been disclosed. In addition, by this invention, as compared to the conventional methods, specific and rapid detection of M. intracellulare in distinction from other Mycobacterium species was enabled.